Smad signalling in the ovary

has now been a decade since the first discovery of the intracellular Smad proteins, the downstream signalling molecules of one of the most important growth factor families in the animal kingdom, the transforming growth factor beta (TGF-beta) superfamily. In the ovary, several TGFbeta superfamily members are expressed by the oocyte, granulosa and thecal cells at different stages of folliculogenesis, and they signal mainly through two different Smad pathways in an autocrine/paracrine manner. Defects in the upstream signalling cascade molecules, the ligands and receptors, are known to have adverse effects on ovarian organogenesis and folliculogenesis, but the role of the individual Smad proteins in the proper function of the ovary is just beginning to be understood for example through the use of Smad knockout models. Although most of the different Smad knockouts are embryonic lethal, it is known, however, that in Smad1 and Smad5 knockout mice primordial germ cell development is impaired and that Smad3 deficient mice harbouring a deletion in exon 8 exhibit impaired folliculogenesis and reduced fertility. In this minireview we discuss the role of Smad structure and function in the ovarian context.has now been a decade since the first discovery of the intracellular Smad proteins, the downstream signalling molecules of one of the most important growth factor families in the animal kingdom, the transforming growth factor beta (TGF-beta) superfamily. In the ovary, several TGFbeta superfamily members are expressed by the oocyte, granulosa and thecal cells at different stages of folliculogenesis, and they signal mainly through two different Smad pathways in an autocrine/paracrine manner. Defects in the upstream signalling cascade molecules, the ligands and receptors, are known to have adverse effects on ovarian organogenesis and folliculogenesis, but the role of the individual Smad proteins in the proper function of the ovary is just beginning to be understood for example through the use of Smad knockout models. Although most of the different Smad knockouts are embryonic lethal, it is known, however, that in Smad1 and Smad5 knockout mice primordial germ cell development is impaired and that Smad3 deficient mice harbouring a deletion in exon 8 exhibit impaired folliculogenesis and reduced fertility. In this minireview we discuss the role of Smad structure and function in the ovarian context.has now been a decade since the first discovery of the intracellular Smad proteins, the downstream signalling molecules of one of the most important growth factor families in the animal kingdom, the transforming growth factor beta (TGF-beta) superfamily. In the ovary, several TGFbeta superfamily members are expressed by the oocyte, granulosa and thecal cells at different stages of folliculogenesis, and they signal mainly through two different Smad pathways in an autocrine/paracrine manner. Defects in the upstream signalling cascade molecules, the ligands and receptors, are known to have adverse effects on ovarian organogenesis and folliculogenesis, but the role of the individual Smad proteins in the proper function of the ovary is just beginning to be understood for example through the use of Smad knockout models. Although most of the different Smad knockouts are embryonic lethal, it is known, however, that in Smad1 and Smad5 knockout mice primordial germ cell development is impaired and that Smad3 deficient mice harbouring a deletion in exon 8 exhibit impaired folliculogenesis and reduced fertility. In this minireview we discuss the role of Smad structure and function in the ovarian context.Peer reviewe

Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are among the key regulators transmitting the signaling between the oocyte and the surrounding granulosa cells. Previously, it has been shown that a recombinant BMP type II receptor ectodomain–Fc fusion protein (BMPR2ecd–Fc) is able to inhibit the actions of GDF9 and BMP15in vitro. Here, we have produced bioactive BMPR2ecd–Fc, which was injected i.p. into neonatal mice. Early folliculogenesis was first studied by injecting mice five times with various doses of BMPR2ecd–Fc during the postnatal days 4–12. Folliculogenesis was affected dose dependently, as evidenced by a decreased mitogenesis of granulosa cells of the growing follicles. Furthermore, we also noticed a decrease in the number of secondary and tertiary follicles as well as an increase in the oocyte size. Electron microscopic analysis revealed that the ultrastructure of the granulosa cells of the primary follicles was not affected by the BMPR2ecd–Fc treatment. A second study was conducted to investigate whether a longer treatment with 12 injections during postnatal days 4–28 would inhibit folliculogenesis. Similar effects were observed in the two studies on the early follicular developmental stages. However, in the long-term study, later stages of folliculogenesis were not blocked but rather increased numbers of antral follicles, preovulatory follicles, and corpora lutea were found. We conclude that BMPR2ecd–Fc is a potent modulator of ovarian folliculogenesisin vivo, and thus, is a valuable tool for studying the physiology and downstream effects of oocyte-derived growth factorsin vivo

Amphiregulin cooperates with bone morphogenetic protein 15 to increase oocyte developmental competence by gap junction-mediated enhanced metabolite supply

This study assessed the participation of amphiregulin (AREG) and bone morphogenetic protein 15 (BMP15) during maturation of bovine cumulus oocyte complexes (COCs) on cumulus cell function and their impact on subsequent embryo development. AREG treatment of COCs enhanced blastocyst formation and quality only when in the presence of BMP15. Expression of hyaluronan synthase 2 was enhanced by follicle stimulating hormone (FSH) but not by AREG, which was reflected in the level of cumulus expansion. Although both FSH and AREG stimulated glycolysis, AREG treated COCs had higher glucose consumption, lactate production and ratio of lactate production to glucose uptake. Autofluorescence levels in oocytes, indicative of NAD(P)H and FAD++, were increased with combined AREG and BMP15 treatment of COCs. In contrast, these treatments did not alter autoflouresence levels when cumulus cells were removed from oocytes, even in the presence of other COCs, suggesting oocyte-cumulus gap-junctional communication (GJC) is required. FSH contributed to maintaining GJC for an extended period of time. Remarkably, BMP15 was equally effective at maintaining GJC even in the presence of AREG. Hence, AREG stimulation of COC glycolysis and BMP15 preservation of GJC may facilitate efficient transfer of metabolites from cumulus cells to the oocyte thereby enhancing oocyte developmental competence. These results have implications for improving in vitro oocyte maturation systems.Satoshi Sugimura, Lesley J Ritter, Melanie L Sutton-McDowall, David G Mottershead, Jeremy G Thompson and Robert B Gilchris

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

TGF-beta mediates proinflammatory seminal fluid signaling in human cervical epithelial cells

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-β1, TGF-β2, and TGF-β3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-β3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-β isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-β induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-β neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-β isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-β in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.David J. Sharkey, Anne M. Macpherson, Kelton P. Tremellen, David G. Mottershead, Robert B. Gilchrist, and Sarah A. Robertso

Signalling pathways involved in the synergistic effects of human growth differentiation factor 9 and bone morphogenetic protein 15

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) act synergistically to regulate granulosa cell proliferation and steroid production in several species. Several non-Sma and mothers against decapentaplegic (SMAD) signalling pathways are involved in the action of murine and ovine GDF9 and BMP15 in combination, with the pathways utilised differing between the two species. The aims of this research were to determine if human GDF9 and BMP15 also act in a synergistic manner to stimulate granulosa cell proliferation and to identify which non-SMAD signalling pathways are activated. Human GDF9 with BMP15 (GDF9 + BMP15) stimulated an increase in 3H-thymidine incorporation (P < 0.001), which was greater than the increase with BMP15 alone, while GDF9 alone had no effect. The stimulation of 3H-thymidine incorporation by GDF9 + BMP15 was reduced by the addition of inhibitors to the SMAD2/3, nuclear factor-KB (NF-KB) and c-Jun N-terminal kinase (JNK) signalling pathways. Inhibitors to the SMAD1/5/8, extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAPK) or p38-MAPK pathways had no effect. The addition of the BMP receptor 2 (BMPR2) extracellular domain also inhibited stimulation of 3H-thymidine incorporation by GDF9 + BMP15. In conclusion, human GDF9 and BMP15 act synergistically to stimulate granulosa cell proliferation, a response that also involves species-specific non-SMAD signalling pathways

Redox and anti-oxidant state within cattle oocytes following in vitro maturation with bone morphogenic protein 15 and follicle stimulating hormone

The developmental competence of cumulus oocyte complexes (COCs) can be increased during in vitro oocyte maturation with the addition of exogenous oocyte-secreted factors, such as bone morphogenetic protein 15 (BMP15), in combination with hormones. FSH and BMP15, for example, induce different metabolic profiles within COCs—namely, FSH increases glycolysis while BMP15 stimulates FAD and NAD(P)H accumulation within oocytes, without changing the redox ratio. The aim of this study was to investigate if this BMP15-induced NAD(P)H increase was due to de novo NADPH production. Cattle COCs were cultured with FSH and/or recombinant human BMP15, resulting in a significant decrease in glucose-6-phosphate dehydrogenase activity (P < 0.05). Inhibition of isocitrate dehydrogenase (IDH) during this process decreased NAD(P)H intensity threefold in BMP15-treated oocytes, suggesting that BMP15 stimulates IDH and NADPH production via the tricarboxylic acid cycle. As NADPH is a reducing agent, reduced glutathione (GSH), H2O2, and mitochondrial activity were also measured to assess the general redox status of the oocyte. FSH alone decreased GSH levels whereas the combination of BMP15 and FSH sustained higher levels. Expression of genes encoding glutathione-reducing enzymes were also lower in oocytes cultured in the presence of FSH alone. BMP15 supplementation further promoted mitochondrial localization patterns that are consistent with enhanced developmental competence. Metabolomics revealed significant consumption of glutamine and production of alanine by COCs matured with both FSH and BMP15 compared to the control (P < 0.05). Hence, BMP15 supplementation differentially modulates reductive metabolism and mitochondrial localization within the oocyte. In comparison, FSH-stimulation alone decreases the oocytes’ ability to regulate cellular stress, and therefore utilizes other mechanisms to improve developmental competence.Fil: Sutton Mcdowall, Melanie L.. University Of Adelaide; Australia. Australian Research Council; AustraliaFil: Purdey, Malcom. Australian Research Council; AustraliaFil: Brown, Hannah. University Of Adelaide; AustraliaFil: Abell, Andrew D.. Australian Research Council; AustraliaFil: Mottershead, David G.. University Of Adelaide; AustraliaFil: Cetica, Pablo Daniel. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Produccion Animal; ArgentinaFil: Dalvit, Gabriel C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Produccion Animal; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Centro de Estudios Transdisciplinarios del Agua; ArgentinaFil: Goldys, Ewa M.. Australian Research Council; Australia. Macquarie University; AustraliaFil: Gilchrist, Robert B.. University Of Adelaide; Australia. University Of New South Wales; AustraliaFil: Gardner, David K.. The University Of Melbourne; AustraliaFil: Thompson, Jeremy G.. University Of Adelaide; Australia. Australian Research Council; Australi

Purification and quantification of pro-mature BMP15.

<p><b>A.</b> Silver stained SDS-polyacrylamide gel of BMP15 samples. The pro-region appeared as two ∼40 KDa bands and the mature region as a ∼16 KDa band and as a ∼17 KDa band that has been reported to be O-linked glycosylated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103563#pone.0103563-Saito1" target="_blank">[68]</a>. Lane 1: flow through. Lanes 2–3: wash 1–2. Lanes 4–7: eluted fractions 1–4. <b>B.</b> The processed mature region of pro-mature BMP15 was quantified by Western blotting [mab28 monoclonal antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103563#pone.0103563-Pulkki1" target="_blank">[42]</a>] using the mature region of hBMP15 (R&D Systems) as a standard. Lanes 1–4: mature region of BMP15 (R&D Systems); 200, 100, 50 and 10 ng, respectively. Lanes 5–7: decreasing doses of the purified BMP15 pro-mature complex.</p